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Journal of the American Mosquito Control Association, 18(4):280-283, 2002 
Copyright © 2002 by the American Mosquito Control Association, Inc. 

THE STRUCTURE AND FUNCTION OF THE LARVAL SIPHON AND 
SPIRACULAR APPARATUS OF COQUILLETTIDIA PERTURBANS 

PETER J. BOSAK 1 and WAYNE J. CRANS 2 

ABSTRACT. The structure of the larval siphon and spiracular apparatus of Coquillettidia perturbans and the 
mechanism of attachment to roots of emergent aquatic macrophytes were examined by utilizing dissection and 
scanning electron microscopy. The roots of these plants contain large air-filled aerenchyma channels that larvae 
of Cq. perturbans pierce with their specialized siphon and spiracular apparatus to breathe. The siphon contains 
the spiracular apparatus, comprising the saw, postabdominal spiracles, inner spiracular teeth, and the spiracular 
apodeme. These are the primary structures that are utilized by larvae to pierce root tissue. Once entry is made 
into a root, the outer spiracular teeth open fully, anchoring the larva in place. 

KEY WORDS Coquillettidia perturbans, cattail mosquito, aerenchyma, siphon, emergent aquatic macrophyte 



INTRODUCTION 

Some of the most remarkable larval respiratory 
adaptations in the family Culicidae occur in the 
genera Mansonia and Coquillettidia. The species of 
these genera have a specialized siphon to pierce the 
roots, stems, or submerged leaves of aquatic plants, 
enabling them to utilize oxygen from plant tissue. 
One species, the cattail mosquito {Coquillettidia 
perturbans (Walker)) is a common nuisance mos- 
quito in North America that has been implicated as 
a bridge vector of eastern equine encephalomyelitis 
virus (Carter et al. 1981, Francy 1982, Sofield et 
al. 1983, Crans and Schulze 1986, Nasci et al. 
1993). Walker described the adult stage of this mos- 
quito in 1 856, naming it Culex perturbans, but be- 
cause of its unusual behavior the larva remained 
undescribed for more than 50 years. The mystery 
was solved in 1907 when J. Turner Brakeley, a vol- 
unteer field observer in New Jersey, washed a 3rd- 
stage larva from the roots of an aquatic grass grow- 
ing in a marsh near Trenton. He reported the find 
to John B. Smith of Rutgers University, who in turn 
published a detailed account of the discovery 
(Smith 1908). Smith's publication incorporated 
John A. Grossbeck's description of the larva and 
several drawings of its morphology, including that 
of the siphon. After Brakeley's discovery, other 
mosquito species that utilized some type of aquatic 
plant as a means for larval respiration were discov- 
ered in other parts of the world (Dyar and Knab 
1910, Ingram and Macfie 1917, Wesenberg-Lund 
1918, Edwards 1919, Gillett 1946, Laurence 1960, 
Burton 1965). Over the years, researchers have ver- 
ified many of Smith's initial observations regarding 
Cq. perturbans; however, apparently no close ex- 
amination was made of the structure and function 
of the siphon and spiracular apparatus. 

In this paper, the morphology of the siphon and 
spiracular apparatus of Cq. perturbans are reex- 



' Cape May County Mosquito Control Commission, PO 
Box 66, Cape May Court House, NJ 08210-0931. 

2 Mosquito Research and Control, Rutgers University, 
180 Jones Avenue, New Brunswick, NJ 08901-8536. 



amined and the mechanism for penetration and at- 
tachment to roots of aquatic macrophytes are de- 
scribed. Harbach and Knight (1980) are followed 
for morphological terminology. 

MATERIALS AND METHODS 

Larval Cq. perturbans were collected at Colliers 
Mills Wildlife Management Area, Colliers Mills, 
NJ, by using the modified bilge pump method of 
Walker and Crans (1986). In the laboratory, 4th- 
stage larvae were isolated and placed in a 250-ml 
beaker with 100 ml of bottled water. Fourth-stage 
larvae were selected because this size enabled the 
clearest view of the siphon and the attachment site. 
Living roots of known host plants were cut from 
masses in lengths of approximately 10-15 cm and 
placed in beakers with several larvae. During the 
warmer months, the setup was left at ambient tem- 
perature, usually overnight, until a number of the 
larvae attached to the roots. During the colder 
months, the setup was placed in refrigeration at 6°C 
overnight. Some larvae would not attach for vari- 
ous reasons, and those that were going to attach 
would usually do so overnight; allowing more time 
did not result in significantly more larval attach- 
ment and may have actually resulted in some de- 
tachment. Once the larvae attached, the setup was 
gradually frozen overnight. With this method, lar- 
vae slowly freeze while remaining attached to the 
roots. Larval Cq. perturbans are very cold tolerant 
and may even withstand freezing for short periods, 
so it is important to freeze the setup overnight. 
Once thawed, small sections of root with attached 
dead larvae were cut free with dissecting scissors, 
carefully removed with forceps, and placed in a pe- 
tri dish containing water. Free-hand cross-section- 
ing of the root with attached larvae was performed 
under a Leica StereoZoom 6 Photo dissecting mi- 
croscope (Leica Microsystems Inc., Buffalo, NY) 
at 40 X. After sectioning, the siphon and the at- 
tached root cross-section were dissected from the 
larva. The prepared sections were placed in a 10% 
NaOH solution and incubated at 35°C overnight for 



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Spiracular Apparatus of Coquillettidia 



281 



clearing and subsequently stained with a weak so- 
lution of Safranine O. 

For scanning electron microscopy, 4th-stage lar- 
vae were collected as above and living specimens 
were air-dried on filter paper. Photographs of struc- 
tural details were taken with a Hitachi® S510 scan- 
ning electron microscope (Hitachi Instruments, San 
Jose, CA) after coating specimens with gold-pal- 
ladium. 



RESULTS 

The siphon and spiracular apparatus (SAp) of 
Cq. perturbans is a modified culicine type, and is 
dark brown and strongly sclerotized. The siphon is 
continuous, lacks sutures, and tapers gradually for 
approximately one half of its length. The apex of 
the siphon is abruptly constricted and bears the an- 
teriorly curved spiracular apparatus that terminates 
sharply. As with other mosquito larvae, Cq. per- 
turbans rely on valves to open and close the SAp 
as needed. When larvae are detached, either swim- 
ming or resting, the valves remain closed and are 
composed of several sclerotized movable plates that 
are relatively smooth (Fig. la). The posterior aspect 
is less complex than the anterior and consists of the 
posterior spiracular plate and posterior spiracular 
lobe (PSL), 2 structures involved in covering the 
spiracular apparatus (Fig. lb). The anterior aspect 
consists of a number of structural elements, the 
most prominent of which is the saw. The sclero- 
tized saw, situated within a furrow in the center of 
the anterior portion of the siphon, is bordered ex- 
ternally by the anterolateral spiracular lobes (LSL) 
(Fig. lc). Internally the saw is fused basally to the 
postabdominal spiracles (PAS) and these in turn are 
joined to the spiracular apodeme (SAd). The PAS 
are 2 fused, rigid tubes that connect with the large 
flexible tracheal trunks in the 8th abdominal seg- 
ment. The SAd is a laterally compressed tubular 
structure that has a strong muscular attachment at 
its proximal end and distally forms a daggerlike 
structure. Lateral to this structure, on each side, are 
triangular plates, and at their apices are the inner 
spiracular teeth (1ST). The 1ST, saw, PAS, and the 
SAd collectively represent the SAp (Fig. 2). 

When the SAp is open, the 1ST and outer spi- 
racular teeth (OST) are fully everted. The 1ST are 
distal to the OST and are situated 1 set on either 
side of the spiracular opening (Fig. 3a). These 
structures are greatly reduced in comparison to the 
OST, are dark in color, and are highly sclerotized. 

The OST are lightly sclerotized and occur in 2 
rows below and lateral to each set of 1ST Each of 
the rows consists of 3 hooklike teeth that are 
stacked one upon the other and when everted curve 
backward and appear to be connected basally to the 
PSL (Fig. 3a). 




Fig. 1. Siphon and spiracular apparatus (closed) of Co- 
quillettidia perturbans. (a) Lateral aspect. LSL, anterolat- 
eral spiracular lobe; PSL, posterolateral spiracular lobe, (b) 
Posterior aspect. S, siphon; PSP, posterior spiracular plate; 
PSL, posterolateral spiracular lobe, (c) Anterior aspect. 
SAW, saw; LSL, anterolateral spiracular lobe. 



DISCUSSION 

Most culicine larvae hang headfirst from the wa- 
ter's surface, venturing below either to feed or es- 
cape danger. Regardless of the time spent sub- 



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Journal of the American Mosquito Control Association 



Vol. 1 8, No. 4 



SAd 




1ST 



PAS 



Fig. 2. Internal structure of the spiracular apparatus of Coquillettidia perturbans. SAd, spiracular apodeme; PAS, 
postabdominal spiracles; SAW, saw; 1ST, inner spiracular teeth. 




Fig. 3. (a) Spiracular apparatus (open) of Coquilletti- 
dia perturbans. OST, outer spiracular teeth; 1ST, inner spi- 
racular teeth; PSL, posterolateral spiracular lobe, (b) Cat- 
tail (Typha latifolia) root cross-section exposing air-filled 
aerenchyma channel pierced by spiracular apparatus of 
Cq. perturbans. 



merged, they must return to the surface to respire 
through a siphon. However, exceptions exist and a 
number of species found in permanent freshwater 
habitats have evolved unique ways to circumvent 
their need to surface for air, and perhaps reduce 
their exposure to predation (McNeel 1 932, Van den 
Assem 1958, Armstrong 1980). Some larvae rely 
on trapped air bubbles in and among vegetation 
whereas others remain submerged by utilizing gill 
or cuticular respiration or both. Coquillettidia per- 
turbans and its near relatives respire by piercing the 
roots of emergent aquatic macrophytes with their 
highly specialized siphons. Emergent aquatic mac- 
rophytes are plants that are at least partly rooted in 
sediment and whose leaves extend into the atmo- 
sphere. The nutrient-rich medium in which these 
plants grow is nearly anoxic, and because roots 
need oxygen to function, some have evolved an 
elaborate gas transport system. Dacey (1981) dem- 
onstrated that aquatic plants such as the yellow wa- 
ter lily (Nuphar luteum) maintain a pressurized 
flow-through ventilation system in which atmo- 
spheric air enters newly unfurled leaves against a 
gradient in pressure and travels down the petioles 
to the rhizomes and roots via a continuous network 
of large open-channeled aerenchyma tissue. As a 
result of the pressure generated by the younger 
leaves, the by-products of root metabolism (carbon 
dioxide and methane) are forced to the atmosphere 
through the plants' older leaves. Compared with the 
roots of terrestrial plants, those of emergent aquatic 
plants contain a greater proportion of aerenchyma 
(Armstrong 1978), and it is these larger air-filled 
channels that larval Cq. perturbans utilize for un- 
derwater respiration. 

In New Jersey, 3 plant species in particular are 
host to larval Cq. perturbans: cattail {Typha spp.), 
rush (Juncus spp.), and swamp loosestrife (Deco- 
don verticillatus) (Crans et al. 1986). Larvae 
searching for an attachment site move along the 
length of the root tapping the epidermis with the 
apex of their siphon, occasionally everting the OST. 
Ingram and Macfie (1917) proposed that fringed 
bristles on the siphon of Mansonia africana (Theo- 
bald) might have a sensory function that aids larvae 



December 2002 



Spiracular Apparatus of Coquillettidia 



283 



in the location of suitable attachment sites. Our ex- 
amination of the siphon and SAp of Cq. perturbans 
did not reveal any fringed bristles and in our opin- 
ion larvae simply test the respiratory suitability of 
the substrate by probing. The depth to which the 
SAp is embedded in root tissue is dependent upon 
its contact with suitable aerenchyma, and very of- 
ten these channels can be found just inside the root 
epidermis (Fig. 3b). Robust muscles attached to the 
SAd provide the strength needed to pierce roots (In- 
gram and Macfie 1917). Penetration of the epider- 
mis is accomplished by rapid, repeated thrusts of 
the terminal segments in concert with the exertion 
of the daggerlike SAd and saw. Once a suitable 
portion of the aerenchyma is penetrated, the 1ST 
and OST open fully. In this position, the OST curve 
backward and anchor the larva in place. This at- 
tachment is a strong one, but larvae can quickly 
release and swim away if disturbed. The 1ST seem 
to play a lesser role in anchoring the larvae and 
may represent a vestigial or ancestral character. 

Larval Cq. perturbans have been assumed and 
widely advocated to possess a siphon equipped 
with an external saw that is used to cut into the 
roots of aquatic plants. Our examination of the si- 
phon and SAp reveals a much more complex struc- 
ture composed of several internal structures that op- 
erate in unison to 1st pierce and then anchor larvae 
into the large air-filled aerenchyma channels found 
in the roots of emergent aquatic macrophytes. 

ACKNOWLEDGMENTS 

We are grateful to Michael May and John La- 
Polla, Department of Entomology, Rutgers Univer- 
sity, for their assistance with the scanning electron 
microscopy, and also James French, Department of 
Plant Science, Rutgers University, for assistance 
with the root dissection. This is New Jersey Agri- 
cultural Experiment Station Publication D-08114- 
14-01 supported by U.S. Hatch Act funds with par- 
tial support from the New Jersey State Mosquito 
Control Commission. 

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